Antioxidative Fish Oil

Research Article

The antioxidative effect of lipophilized rutin anddihydrocaffeic acid in fish oil enriched milk

Ann-Dorit Moltke Sørensen1, Lone Kirsten Petersen1*, Sara de Diego2**, Nina Skall Nielsen1,

Bena-Marie Lue3***, Zhiyong Yang3, Xubing Xu3 and Charlotte Jacobsen1

1 Division of Industrial Food Research, National Food Institute, Technical University of Denmark, Lyngby,

Denmark2 Department of Biotechnology and Food Science, University of Burgos, Burgos, Spain3 Department of Engineering, Aarhus University, Arhus, Denmark

The antioxidative effect of phenolipids was evaluated in fish oil enriched milk emulsions as a model for a

complex food system. Two different phenolipids modified from dihydrocaffeic acid (with C8 or C18:1) and

rutin (with C12 or C16) were evaluated. Both dihydrocaffeate esters and rutin laurate showed significantly

better antioxidant properties inmilk emulsion compared with the original phenolics. However, rutin palmitate

only performed slightly better as antioxidant than rutin. The results with rutin indicated that a cut-off effect

exists in relation to the alkyl chain length with respect to optimal antioxidant activity in milk emulsions. Thus,

the optimal alkyl chain length is at least below 16 carbon atoms, and maybe even less for rutin esters. For

dihydrocaffeate esters it was not possible to conclude on a cut-off effect in relation to alkyl chain length and

antioxidative effect due to the almost similar antioxidant effect of the two phenolipids. However, there was a

tendency towards octyl dihydrocaffeate being slightly more efficient than oleyl dihydrocaffeate.

Practical application: The finding that phenolipids are better antioxidants in milk emulsions than the

original phenolic acid provides new knowledge that can be used to develop new antioxidant strategies to

protect foods against lipid oxidation. However, the results indicate that both optimization of alkyl chain

length for each type of phenolic, and optimization for each type of emulsion will be necessary in order to get

the best oxidative stability of an emulsion with these phenolipids. Use of efficient antioxidantsmay lower the

amount of antioxidant needed to protect against lipid oxidation and may in addition decrease the costs.

Keywords: Caffeic acid / o/w Emulsion / Polar paradox / Rutin

Received: October 10, 2011 / Revised: January 9, 2012 / Accepted: February 24, 2012

DOI: 10.1002/ejlt.201100354

1 Introduction

The health beneficial effects of n-3 long chain PUFA (LC

PUFAs) such as, e.g. reduced risk of cardiovascular diseases

are well documented. During the last decade substantial

efforts have been put into enriching foods with the healthy

n-3 LC PUFAs as reviewed by Jacobsen et al. [1] These

efforts have been carried out in order to increase the popu-

lations’ intake of especially eicosapentaenoic acid (EPA) and

DHA [2, 3]. Despite the increasing number of n-3 PUFA

enriched foods on the market, consumer acceptance and

shelf-life of such products are still limited by the higher

oxidative susceptibility of unsaturated lipids, which will lead

to an unpleasant fishy off-flavour [4–7]. To retard lipid oxi-

dation, a range of commercial synthetic antioxidants with free

radical scavenging activity and metal chelating properties are

*Current address: CP Kelco ApS, Ved banen 16, DK-4623 Lille Skensved,

Denmark

**Current address: Grupo Siro, Paseo Pintor Rosales 40, Madrid, Spain

***Current address: Novozymes A/S, Krogshoejvej 36, DK-2880

Bagsvaerd, Denmark

Correspondence: Dr. Ann-Dorit Moltke Sørensen, Division of Industrial

Food Research, National Food Institute, Technical University of Denmark,

Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark

E-mail: [email protected]

Fax: þ45 4588 4774

Abbreviations: ATD, automatic thermal desorber; BHT, butylated

hydroxytoluene; DHCA, dihydrocaffeic acid; DHCA C18:1, oleyl

dihydrocaffeate; DHCA C8, octyl dihydrocaffeate; EPA,

eicosapentaenoic acid; LC, long chain; PV, peroxide value; Rutin C12,

rutin laurate; Rutin C16, rutin palmitate

434 Eur. J. Lipid Sci. Technol. 2012, 114, 434–445

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

available, e.g. calcium disodium ethylenediaminetetraacetate

(EDTA), butylhydroxytoluene (BHT) and propyl gallate.

However, there is a trend in consumer preference for natural

ingredients such as phenolic compounds rather than syn-

thetic compounds. The major part of natural antioxidants

from plants, are phenolic compounds, e.g. caffeic acid.

Most food products are emulsions, and in these systems

lipid oxidation is suggested to be initiated at the interface

between the oil phase and the aqueous phase or air, and

continued in the oil phase. In emulsions, antioxidants may

mainly partition into three different phases: the aqueous

phase, the oil phase and the interface between oil and water.

Partitioning of antioxidants into the different phases is influ-

enced by their polarity and interactions with other com-

ponents present in the emulsion, e.g. emulsifier [8, 9].

Generally, phenolics are hydrophilic compounds and they

will most likely be located in the aqueous phase.

Furthermore, the polarity of antioxidants in bulk oil and

emulsions has been considered to be decisive for their effi-

ciency. This phenomenon is known as the polar paradox and

states that hydrophilic antioxidants are more efficient in bulk

oils than lipophilic antioxidants. In contrast, lipophilic anti-

oxidants generally function better than hydrophilic anti-

oxidants in emulsions [10]. However, recent studies have

reported results that contradict the polar paradox hypothesis

[11–14]. This suggests that other factors might be equally

important, and more research is urgently required to improve

our understanding about the relationship between the mol-

ecular structure of the antioxidants and their efficacy in

different real food systems.

Recently, several studies have reported the possibility of

changing the polarity of phenolics by lipophilization with fatty

acids of different chain length in order to improve their

antioxidative effect in emulsified media. The current work

in this area has been summarized by Shahidi and Zhong [15].

Laguerre et al. [11, 16] have recently reported on the anti-

oxidative effect of lipohilized chlorogenic and rosmarinic

acids. For chlorogenic acid the antioxidant capacity increased

as the alkyl chain length was increased from 1 to 12 carbon

atoms, whereas further increase of the chain length resulted in

a drastic decrease in the antioxidant capacity. On the basis of

these results the authors suggested a so-called cut-off effect

related to the length of the lipid chain attached to chlorogenic

acid, which they explained as follows: When, the hydropho-

bicity of the lipophilized compound increases to above a

certain level, the lipophilized compound is suggested to form

micelles in the aqueous phase. Thereby, they will not be

available as antioxidants at the interface and in turn their

efficacy will be reduced [11]. For rosmarinate esters, the octyl

rosmarinate improved the antioxidative effect eight times

compared to rosmarinic acid. Thus, the results lead to the

conclusion that lipophilization with medium chain fatty acids

is a promising way to increase the antioxidant activity [16].

However, the results obtained with chlorogenate and ros-

marinate esters have also led to the suggestion that the exact

location of antioxidants in the discontinuous phase, inter-

facial layer or oil droplets, is important for the activity of the

antioxidants [11, 16]. In addition, a study on lipophilized

dihydrocaffeic acids and their antioxidative effect in o/w

emulsions reported by Sørensen et al. [17] suggested that

lipophilized dihydrocaffeic acid tended to follow the newly

suggested cut-off effect in relation to the alkyl chain length

attached although only two chain lengths were evaluated. In

contrast, lipophilized rutin added to o/w emulsions did not

show a cut-off effect, since the esters, rutin laurate and rutin

palmitate, were consistently less effective compared with the

rutin [13]. However, only two chain lengths (C12 and C16)

were evaluated along with rutin, hence alkyl chain shorter

than C12 lipophilized on rutin may indicate a cut-off effect.

From these studies it may be concluded that the cut-off effect

is specific for the individual lipophilized phenolic com-

pounds, i.e. the optimal chain length may vary between

different phenolics.

Recent research on lipophilized phenolic compounds has

mostly paid attention to their production, in vitro antioxidant

activity and their effect in simplified model systems, whereas

studies on their effect in real food systems are lacking. Inmore

complex systems such as real food the antioxidant behaviour

might be influenced by interaction with other components

present in the emulsions, e.g. emulsifier and iron [15, 18].

However, on the basis of studies in simple o/w emulsions we

hypothesize that lipophilization of phenolics will increase

their antioxidant efficacy in emulsified food enriched with

n-3 PUFAs. Moreover, we hypothesize that the alkyl chain

length will affect the antioxidative effect of these lipophilized

phenolic compounds as was observed in a recent study with

simple o/w emulsions [17], and that the effect of the alkyl

chain length may be different from that observed in simple

model systems due to the presence of other potentially inter-

acting compounds. Therefore, the aim was to evaluate the

antioxidative effect of dihydrocaffeic acid lipophilized with

either octyl or oleyl alcohol and rutin lipophilized with lauric

acid or palmitic acid in fish oil enriched milk. Fish oil

enriched milk was chosen as previous studies have shown

that it is highly susceptible to lipid oxidation [1]. Moreover, it

is a complex food system in which antioxidants may interact

with different compounds, e.g. proteins. Lipophilized rutin

and dihydrocaffeic acid were chosen as antioxidants since this

study is a continuation of recent studies performed in our lab

in simplified o/w emulsions [12, 13, 17] and results could

show different effects of lipophilization in simple versus com-

plex emulsions.

2 Material and methods

2.1 Materials

Fresh milk (0.5 and 1.5% fat content) was purchased in a

local supermarket. Fish oil without antioxidant added was

supplied by Maritex Norway (subsidiary of TINE BA,

Eur. J. Lipid Sci. Technol. 2012, 114, 434–445 Antioxidant efficacy of lipophilized phenolics 435


Norway). This oil had an initial PV of 0.1 meq peroxides/kg

oil, tocopherol content of 204 mg a-tocopherol, 102 mg g-

tocopherol and 42 mg d-tocopherol/kg oil, and the fatty acid

composition was as follows: 14:0, 3.0%; 16:0, 8.7%; 16:1,

8.2%; 18:0, 1.9%; 18:1, 20.9%; 18:2, 1.8%; 18:4, 2.6%;

20:1, 12.5%; 20:5 (EPA), 9.4%; 22:1, 5.9%; 22:5, 1.1%

and 22:6 (DHA), 11.6%. The total percentages of n-3 and

n-6 PUFA in the oil were 24.7 and 2.7%, respectively.

Rutin (purity �98%), caffeic acid (purity �98%),

dihydrocaffeic acid (purity �98%) and oleyl alcohol (purity

85%) were from Sigma–Aldrich (Steinheim, Germany).

Lipophilized rutin with lauric (C12) or palmitic (C16) acids,

both with a purity of 98%, were synthesized at the National

Food Institute, Division of Industrial Food Research

(Technical University of Denmark, Lyngby Denmark). For

further details about the lipophilization process refer to Lue

et al. [19]. Lipophilized dihydrocaffeic acid with octyl (C8)

with a purity of 80% or oleyl alcohol (C18:1) with a purity of

60% were synthesized at the Department of Engineering,

Faculty of Science (Aarhus University, Arhus, Denmark).

Chemicals were from Merck (Darmstadt, Germany) and

external standards for identification and quantification of

secondary volatile oxidation products were all from Sigma–

Aldrich (Steinheim, Germany). All solvents were of HPLC

grade and purchased from Lab-Scan (Dublin, Ireland).

2.2 Production of fish oil enriched milk

Milk with 0.5% fat and with 1.5% fat was mixed (1:1 v/v) to

obtain a total fat content of 1%. Subsequently, the milk was

pasteurized at 728C for 15 s and the fish oil (0.5% v/v) and

antioxidant were added (for specification on antioxidant

addition, see Section 2.3). Emulsions were prepared in two

steps: pre-emulsification and homogenization. During pre-

emulsification, the heated milk with fish oil and antioxidant

added was stirred with an Ultra-Turrax (Step 7, 1 min, Janke

& Kunkel IKA-Labortechnik, Staufen, Germany). The pre-

emulsion was then homogenized at a pressure of 25 and

250 bar with four circulations of the emulsion at RT using

a table homogenizer from GEA Niro Soavi Spa (Parma,

Italy). The emulsions (100 g) were stored in 100 mL blue

cap bottles at 58C. Samples, one flask pr. code, were taken at

Day 0, 3, 6, 9 and 12 and divided in brown glass bottles for

different analysis and stored at �408C until analyses of per-

oxides, volatiles, tocopherols and fatty acids were performed.

The droplet size was measured at Day 1, 6 and 12 without

pre-freezing.

2.3 Experimental design

The experimental design for Experiment 1 and 2 is described

in details below and summarized in Table 1.

Experiment 1: Five different antioxidants were evaluated,

rutin, rutin laurate, rutin palmitate, dihydrocaffeic acid and

oleyl dihydrocaffeate in a concentration of 100 mM in fish oil

enriched milk. Dihydrocaffeic acid and oleyl dihydrocaffeate

were added directly to the milk, whereas rutin, rutin laurate

and rutin palmitate were first dissolved or suspended in

1.5 mL acetone due to dissolving problems in the milk emul-

sion. The acetone with antioxidant was then added to the

heated milk. To obtain the same condition for all emulsions,

1.5 mL acetone was added to the milk emulsions with dihy-

drocaffeic acid, oleyl dihydrocaffeate and the control (no

antioxidant added).

Experiment 2: Four different antioxidants were evaluated,

caffeic acid, dihydrocaffeic acid, octyl dihydrocaffeate and

oleyl dihydrocaffeate. Antioxidant concentration tested was

similar to that in Experiment 1 (100 mM). The synthesized

Table 1. Experimental design

Antioxidant applied Sample code

Concentration of antioxidant

mM mg/kg

Experiment 1: Acetone addition (1.5 mL)

Control Control – –

Rutin Rutin 100 61.1

Rutin laurate (C12) Rutin C12 100 79.3

Rutin palmitate (C16) Rutin C16 100 84.9

Dihydrocaffeic acid DHCA 100 18.2

Oleyl dihydrocaffeate (C18:1) DHCA C18:1 100 72.1

Experiment 2: No acetone addition

Control Control – –

Dihydrocaffeic acid DHCA 100 18.2

Octyl dihydrocaffeate (C8) DHCA C8 100 36.8

Oleyl dihydrocaffeate (C18:1) DHCA C18:1 100 72.1

Oleyl alcohol Oleyl alcohol 107 28.8

Caffeic acid Caffeic acid 100 18.0

436 A.-D. M. Sørensen et al. Eur. J. Lipid Sci. Technol. 2012, 114, 434–445


oleyl dihydrocaffeate was only 60% pure, and contained

�40% free oleyl alcohol. Therefore, an emulsion with oleyl

alcohol with the same amount of oleyl alcohol (29 mg/kg) as

in the emulsion with oleyl dihydrocaffeate was included to

evaluate the effect of oleyl alcohol on lipid oxidation in the

milk emulsion. The antioxidants were dissolved directly in

the heated milk before homogenization.

2.4 Droplet size determination

The size of the fat droplets in milk emulsions was determined

by laser diffraction with a Mastersizer 2000 (Malvern

Instruments Ltd., Worcestershire, UK). A few droplets of

the milk emulsion were suspended directly in re-circulating

water (2800 rpm, obscuration 14–16%). The set-up used

was the Fraunhofer method, which assumes that all sizes

of particles scatter with equal efficiencies and that the

particles is opaque and transmits no light [20]. The results

were reported as surface mean diameter,

D3;2 ¼P

nid3iP

nid2i

where d is the diameter of individual droplets.

2.5 Measuring lipid oxidation

2.5.1 Extraction of lipids

Lipids were extracted from fish oil enriched milk (15 g)

according to the method described by Bligh and Dyer [21]

with reduced amount of solvent applied [22]. The analysis

was done in duplicate and further used for determination of

peroxide value, fatty acid composition and tocopherol

concentration.

2.5.2 Primary oxidation products, peroxide value (PV)

Peroxide value in the lipid extracts were determined by a

colorimetric method based on formation of an iron–thiocya-

nate complex measured according to the method described

by Shanta and Decker [23], n ¼ 2.

2.5.3 Secondary volatile oxidation products –dynamic headspace

Volatiles were collected on TenaxTM tubes (Perkin Elmer,

Norwalk, CT, USA) by purging the fish oil enriched milk

(8 g) with nitrogen (150 mL/min, 30 min) at 458C. An

ATD-400 automatic thermal desorber was used for thermally

desorbing the collected volatiles. The transfer line of the

ATD was connected to a 5890 IIA gas chromatograph

(Agilent Technologies, Palo Alto, CA, USA) equipped with

a DB wax column (length 30 m � I.D. 0.25 mm � 0.5 mm

film thickness, J&W Scientific, CA, USA) coupled to a HP

5972A mass selective detector. Temperature program was as

follows: 5 min at 458C, 1.58C/min from 45 to 558C,

2.58C/min from 55 to 908C, 128C/min from 90 to 2208Cand hold for 4 min at 2008C. The MS was operating in the

electron ionization mode at 70 eV and mass to charge ratios

between 29 and 200 were scanned. For quantification of the

different volatiles, solutions with external standards at differ-

ent concentrations were prepared and analysed from milk

with no fish oil added. The results are given in ng/g milk

(n ¼ 3).

2.5.4 Tocopherol concentration

Lipid extract was evaporated under nitrogen, re-dissolved in

heptane and analysed byHPLC (Agilent 1100 Series, Agilent

Technologies, Palo Alto, CA, USA) according to the AOCS

method [24] to determine tocopherol concentration in the

different milk samples (n ¼ 4).

2.5.5 Fatty acid composition

Lipid extract was evaporated under nitrogen. First, the glyc-

erol bound fatty acids were transesterified with methanolic

NaOH (0.5 M). Then, hydrolytic released and free fatty acids

were methylated by a boron trifluoride reagent (20%) cata-

lysed process. Methyl esters were extracted with heptane

followed by separation on GC (HP 5890A, Agilent

Technologies, Palo Alto, CA, USA). The procedure was

according to the AOCS methods [25, 26], n ¼ 2.

2.5.6 Sensory

Preliminary sensory evaluation was performed by an expert

panel composed of two persons. Each assessor evaluated one

milk sample at a time. After each milk sample the assessors

discussed the odour of the sample (only Experiment 2).

2.6 Data analyses

2.6.1 Statistics

The obtained results were analysed by two way ANOVA

(GraphPad Prism, Version 4.03, GraphPad Software,

Inc.). The Bonferroni multiple comparison was used to test

differences between samples or storage time (significance

level p<0.05).

2.6.2 Inhibition percentages

To compare the efficacy of the antioxidants in the two differ-

ent emulsion systems, inhibition percentages were calculated.

Inhibition ½%� ¼ 1� SampleAntioxidantSampleControl

� �� 100



3 Results

3.1 Characteristics of the fish oil enriched milkemulsions

The characteristics, i.e. droplet size and content of EPA and

DHA of the different fish oil enriched milk emulsions are

summarized in Table 2. Average sizes of the lipid droplet in

the milk were between 0.45 and 0.74 mm during the storage

period. In Experiment 1, the emulsion with antioxidants had

a significantly larger droplet size, whereas a similar effect was

not observed in the second experiment. The droplet sizes

measured in the different milk emulsion were unchanged

during storage. Content of EPA and DHA in the different

milk emulsions at day 0 indicated similar levels of 3.26–3.47

and 4.03–4.44% EPA and DHA of total fatty acids, respect-

ively. At Day 12, oleyl dihydrocaffeate (Experiment 2) was

the only milk emulsion with a decrease in EPA and DHA

>0.1%. However, the EPA and DHA contents increased to

the same extent during storage in several emulsions. Thus,

the decreases in EPA and DHA in oleyl dihydrocafeate

(Experiment 2) may be due to day to day variation for the

measurements rather than an actual decrease. This finding

suggests that lipid oxidation did not significantly affect EPA

and DHA contents in any of the milk emulsions.

3.2 Peroxide values in fish oil enriched milkemulsions

Peroxide values obtained in Experiment 1 and 2 during

storage are shown in Fig. 1. For Experiment 1 a lag phase

Table 2. Droplet size D3,2 [mm] for lipid droplets in fish oil enriched milk given as an average during storage (average W SD) and content of

EPA and DHA (wt% of total lipids) at day 0 and 12 in the different milk emulsions

Sample code Droplet size (mm)

EPA content (%wt of total lipids) DHA content (%wt of total lipids)

Day 0 Day 12 Day 0 Day 12

Experiment 1

Control 0.45 � 0.01 3.47 � 0.08 3.51 � 0.09 4.35 � 0.24 4.47 � 0.21

Rutin 0.70 � 0.03 3.26 � <0.01 3.51 � 0.15 4.03 � 0.07 4.43 � 0.28

Rutin C12 0.74 � 0.01 3.42 � 0.04 3.48 � <0.01 4.26 � 0.20 4.30 � 0.06

Rutin C16 0.59 � 0.01 3.43 � 0.08 3.44 � 0.01 4.26 � 0.21 4.27 � 0.01

DHCA 0.70 � 0.03 3.47 � 0.05 3.58 � 0.01 4.35 � 0.15 4.57 � <0.01

DHCA C18:1 0.72 � 0.02 3.51 � 0.08 3.48 � 0.01 4.44 � 0.16 4.36 � 0.01

Experiment 2

Control 0.73 � 0.02 3.41 � 0.01 3.75 � 0.07 4.15 � 0.03 4.53 � 0.02

DHCA 0.65 � 0.07 3.37 � 0.02 3.31 � 0.04 4.10 � 0.04 4.04 � 0.08

DHCA C8 0.70 � 0.03 3.31 � 0.01 3.31 � 0.07 4.05 � 0.04 4.02 � 0.16

DHCA C18:1 0.74 � 0.04 3.43 � 0.08 3.26 � 0.06 4.09 � 0.01 3.99 � 0.02

Oleyl alcohol 0.73 � 0.02 3.46 � 0.01 3.58 � 0.01 4.22 � 0.04 4.45 � 0.03

Caffeic acid 0.61 � 0.09 3.37 � 0.07 3.37 � 0.02 4.13 � 0.09 4.07 � 0.04

Sample codes refer to Experimental design Table 1.

0 5 10 150

5

10

15

Storage time [Days]

PV [m

eq p

erox

ides

/ kg

oil]

0 5 10 150

1

2

3

4

5

6

7

PV [m

eq p

erox

ides

/ kg

oil]

(A)

(B)

ControlRutin palmitate

Rutin laurate

RutinOleyl dihydrocaffeateDihydrocaffeic acid

Oleyl alcoholControl

Dihydrocaffeic acidCaffeic acid

Oleyl dihydrocaffeateOctyl dihydrocaffeate

Figure 1. Concentration of peroxides measured as PV [meq.

peroxides/kg oil] in the different fish oil enriched emulsions during

storage. Error bars indicate SD of the measurements (n ¼ 2). (A)

Experiment 1: control (&), rutin (D), rutin laurate (~), rutin palmitate

(!), dihydrocaffeic acid (*) and oleyl dihydrocaffeate (*). (B)

Experiment 2: control (&), dihydrocaffeic acid (*), octyl dihydro-

caffeate (^), oleyl dihydrocaffeate (*), oleyl alcohol (X) and caffeic

acid (5).



was observed until day 3 for the emulsion without antioxidant

added (Fig. 1A) as the difference between day 0 and 3 was

insignificant. In contrast, the lag phase for emulsion with

antioxidant added was longer than 3 days and for milk emul-

sion with rutin laurate the lag phase could not be determined

within the storage period (PV never increased). This indi-

cated that rutin laurate efficiently inhibited the development

of peroxides. However, the data could also indicate that

peroxides were developed and decomposed at equal rates.

Investigation of secondary oxidation products (volatiles),

described later, resolves this. After the end of the lag phase,

the concentration of peroxides increased with different rates

in the different emulsions. At day 12 the ranking of the different

milk emulsions with respect to PV level was as follows: controla

(highest level) � rutin palmitateab � rutinb ¼ oleyl dihydro-

caffeateb ¼ dihydrocaffeic acidb>rutin lauratec.

Compared to Experiment 1, the off set for peroxide devel-

opment was faster in Experiment 2 (Fig. 1B). Moreover, the

PV level in the milk emulsion without antioxidant and

the one with dihydrocaffeic acid added was higher than in

the samemilk emulsion in Experiment 1. Only two emulsions

in Experiment 2 had a lag phase, and these were the milk

emulsions with oleyl dihydrocaffeate and octyl dihydrocaf-

feate added. For all other milk emulsions the development of

PVs was triggered in different rates already at the beginning of

storage depending on the antioxidant added. At day 12 the

ranking ofmilk emulsions according to PV level was as follows:

oleyl alcohola>controlb>dihydrocaffeic acidc ¼ caffeic

acidc>oleyl dihydrocaffeated ¼ octyl dihydrocaffeated.

Thus, in contrast to the findings for rutin esters in

Experiment 1, the chain length of the fatty acid esterified

to dihydrocaffeic acid did not influence the development of

peroxides in the emulsions differently.

The inhibition percentage calculated for Experiment 1

based on PV development (Table 3), clearly showed that

rutin laurate was the most efficient antioxidant in inhibiting

the formation of peroxides when added to milk emulsion.

However, oleyl dihydrocaffeate and rutin palmitate was as

efficient as rutin laurate at Day 6, but their antioxidative

efficiency decreased after Day 6. For Experiment 2 it is clear

from the inhibition percentage that both dihydrocaffeate

esters added were very effective in inhibiting the peroxides

formation. Inhibition percentages for those milk emulsions

with the same antioxidants applied in both experiments;

dihydrocaffeic acid and oleyl dihydrocaffeate, indicated bet-

ter inhibition of PV development in Experiment 1 with

acetone added for dihydrocaffeic acid emulsion after Day 3

and in Experiment 2 without acetone for oleyl dihydrocaf-

feate emulsion. Thus, the addition of acetone seemed to have

no significant impact on the development of peroxides. In

spite of the higher PVs in Experiment 2, the efficacy of

antioxidants in fish oil enriched milk seemed to be better

for octyl and oleyl dihydrocaffeate without acetone than

oleyl dihydrocaffeate, rutin laurate and rutin palmitate with

acetone added at least before Day 9. However, before a

clear conclusion can be drawn the data on volatile oxidation

products must be considered.

3.3 Development of volatile oxidation products

Five different volatiles were measured in the stored milk

emulsions and three of them is shown in Fig. 2: 1-penten-

3-one, 1-penten-3-ol and 2,6-nonadienal. These volatiles are

shown since they illustrate the general trend in the develop-

ment of volatiles during storage in the two experiments.

Furthermore, they represent decomposition of n-3 fatty acids

and are known to have impact on the development of fishy

off-flavour [27].

For Experiment 1, the volatiles are shown in Fig. 2A, C

and E, respectively. Concentration of 1-penten-3-one in milk

Table 3. Calculated inhibition percentages of PV level in the different milk emulsions in both storage experiments

Milk emulsions

Inhibition of PV during storage (%)

Day 0 Day 3 Day 6 Day 9 Day 12

Experiment 1

Rutin �6 18 42 34 21

Rutin laurate (C12) 27 32 59 77 78

Rutin palmitate (C16) 37 �18 57 32 11

Dihydrocaffeic acid 18 �15 31 52 29

Oleyl dihydrocaffeate (C18:1) 34 21 58 31 26

Experiment 2

Dihydrocaffeic acid 35 29 11 15 23

Octyl dihydrocaffeate (C8) 45 63 83 57 57

Oleyl dihydrocaffeate (C18:1) 47 59 70 59 49

Oleyl alcohol 63 3 �95 26 �15

Caffeic acid 57 51 33 34 31

The values are calculated according to the control emulsions from the respective experiment.



emulsions increased until Day 9, whereafter the concen-

tration decreased at different rates in the different emulsions.

Milk emulsions with no antioxidant and dihydrocaffeic acid

added had no lag phase, whereas milk emulsions with rutin,

rutin palmitate and oleyl dihydrocaffeate added had a lag

phase of 3 days before these emulsions started developing 1-

penten-3-one. The concentration of 1-penten-3-one in milk

emulsion with rutin laurate added did not increase during

storage. The ranking of the emulsions according to concen-

tration of 1-penten-3-one before the decrease in concentration

(Day 9) was as follows: rutin lauratea<oleyl dihydrocaffea-

teb<rutin palmitatec � dihydrocaffeic acidc<rutind<controle.

The observed decline in the concentration of 1-penten-3-one at

the end of the storage period might be due to a reduction of

this volatile to 1-penten-3-ol either by the antioxidant or

other components in the milk emulsion. For the other vol-

atiles measured in this experiment, the concentration

increased after a shorter or longer lag phase (Fig. 2C

and E). Similar for these volatiles were that the control milk

emulsion had the shortest lag phase and the highest concen-

tration of the volatiles measured at the end of the storage. The

lag phase for the development of 1-penten-3-ol in milk emul-

sion was 3 days for control emulsion and emulsions with

dihydrocaffeic acid, and 6 days for oleyl dihydrocaffeate,

rutin and rutin palmitate added, whereas milk emulsion with

rutin laurate had a lag phase of 9 days (Fig. 2C). The duration

of the lag phase for development of 2,6-nonadienal was

slightly different from that of 1-penten-3-ol. Here, the milk

emulsion with rutin only had 3 days lag phase together with

dihydrocaffeic acid and control emulsions, andmilk emulsion

with oleyl dihydrocaffeate added had an infinite lag phase

(2,6-nonadienal never increased, Fig. 2E). After the end of

the lag phase the concentration of volatiles increased in the

different milk emulsions, except for 2,6-nonadienal in the

Figure 2. Concentration of three volatiles [ng/g] in the different fish oil enriched milk emulsions during storage. (A,B) 1-penten-3-one

Experiment 1 and 2, (C,D) 1-penten-3-ol Experiment 1 and 2, (E,F) 2,6-nonadienal Experiment 1 and 2, respectively. Error bars indicate

SD of the measurements (n ¼ 3). Symbols: control (&), rutin (D), rutin laurate (~), rutin palmitate (!), dihydrocaffeic acid (DHCA*), oleyl

dihydrocaffeate (DHCA C18:1 *), octyl dihydrocaffeate (DHCA C8 ^), oleyl alcohol (X) and caffeic acid (5).



emulsion with oleyl dihydrocaffeate added. At day 12 the

ranking according to concentration of 1-penten-3-ol and 2,6

nonadienal were as follows: rutin lauratea<oleyl dihydrocaf-

feate and dihydrocaffeic acidb<rutin and rutin palmita-

tec<controld and oleyl dihydrocaffeatea<rutin laurate and

dihydrocaffeic acidb<rutin palmitate and rutinc<controld,

respectively. Overall, the concentrations of volatiles were

lower in emulsions containing the lipophilized compounds

compared with the original phenolic. However, rutin palmi-

tate sometimes led to higher concentration of volatiles than

rutin in the milk emulsions.

The development of volatiles in emulsions from

Experiment 2 is shown in Fig. 2B, D and F. As observed

in Experiment 1, the concentration of 1-penten-3-one first

increased and then decreased. However, in Experiment 2 this

only happened in three emulsions: oleyl alcohol, dihydrocaf-

feic acid and control emulsion. Moreover, these emulsions

had a faster increase in the concentration of 1-penten-3-one

than the other emulsions. For milk emulsions with octyl and

oleyl dihydrocaffeate added, the development of 1-penten-3-

one did not increase the first 3 days. The concentration of 1-

penten-3-one in the emulsions before the decrease (Day 6)

was ranked as follows: octyl dihydrocaffeatea<oleyl dihydro-

caffeatea,b<caffeic acidb<dihydrocaffeic acidc<controld<oleyl

alcohole (Fig. 2B). The same milk emulsions, as observed for

development of 1-penten-3-one, also had a faster increase for

the other measured volatiles, except for 2,6-nonadienal. In

these emulsions (control, dihydrocaffeic acid and oleyl alco-

hol) no lag phase was observed, whereas the other 3 emul-

sions with caffeic acid, oleyl dihydrocaffeate and octyl

dihydrocaffeate added, had a lag phase. For the development

of 1-penten-3-ol, the lag phase was 3 days for milk emulsion

with caffeic acid and oleyl dihydrocaffeate added, whereas it

was 9 days with octyl dihydrocaffeate added. At the end of the

storage period the concentration of 1-penten-3-ol in the

different emulsions was ranked as follows: octyl dihydrocaf-

featea<oleyl dihydrocaffeateb<caffeic acidc<dihydrocaffeic

acidd<controle<oleyl alcohold. Similar to Experiment 1,

the development of 2,6-nonadienal had a longer lag phase

than observed for the other volatiles measured. Emulsions

with oleyl alcohol and dihydrocaffeic acid had together with

the control emulsion, the shortest lag phases of 3 days fol-

lowed by the emulsion with caffeic acid (6 days). The longest

lag phase was observed in emulsions with octyl and oleyl

dihydrocaffeate, which continued during the entire storage

period (Fig. 2F). According to concentration of 2,6-nona-

dienal at Day 12, the order of emulsions was as follows: oleyl

and octyl dihydrocaffeatea<caffeic and dihydrocaffeic acid-

sb<controlc<oleyl alcohold. In summary, the difference for

the different volatiles was the length of the lag phase, whereas

the ranking of the emulsions according to volatile concen-

trations was more or less similar for the different volatiles.

Similar to the PV, the inhibition percentages were calcu-

lated, however only at the end of the storage and for the

concentration of 1-penten-3-one when it was highest at day 9

and 6 for Experiment 1 and 2, respectively. The inhibition

percentage from Experiment 1 (Table 4) clearly showed that

rutin laurate followed by oleyl dihydrocaffeate were the most

efficient antioxidants in inhibiting the formation of volatiles

compared to other antioxidants. Depending on the volatile, it

was different whether it was rutin or rutin palmitate that was

more efficient. For 1-penten-3-ol and 2,4-heptadienal rutin

was more efficient compared to rutin palmitate, whereas

for 1-penten-3-one, 2-hexenal and 2,6-nonadienal it was

Table 4. Calculated inhibition percentages of volatile concentration in the different milk emulsions in both storage experiments

Milk emulsions

Inhibition of volatiles at day 12 (%)

1-Penten-3-onea) 1-Penten-3-ol 2-Hexenal 2,4-Heptadienal 2,6-Nonadienal

Experiment 1

Rutin 17 48 22 25 25

Rutin laurate 76 78 48 63 65

Rutin palmitate 39 41 26 14 34


Oleyl dihydrocaffeate 52 66 41 60 b)

Experiment 2

Caffeic acid 69 45 31 32 47


Oleyl dihydrocaffeate 75 66 43 54 b)

Octyl dihydrocaffeate 80 77 54 46 b)

Oleyl alcohol �13 �26 �25 �7 �55

The values were calculated according to the control emulsions from the respective experiment.a) For development of 1-penten-3-one the inhibition percentages were calculated at the day with highest concentration before decreasing, that

means that it was calculated at day 9 for experiment 1 and day 6 for experiment 2.b) This indicate that the specific volatile was not detected in this emulsion and therefore the inhibition percentage was not calculated.



opposite. For Experiment 2 it is clear from the inhibition

percentages that both dihydrocaffeate esters were very effec-

tive in inhibiting the formation of volatiles. However, octyl

dihydrocaffeate was more efficient regarding the inhibition of

1-penten-3-one, 1-penten-3-ol and 2-hexenal, whereas oleyl

dihydrocaffeate was more efficient in inhibiting the formation

of 2,4-heptadienal. When comparing the efficiency of the

phenolics, caffeic acid was more efficient as antioxidant than

dihydrocaffeic acid. The impurity, oleyl alcohol, clearly

showed prooxidative behaviour in the milk emulsion.

Inhibition percentages in milk emulsions with the same anti-

oxidants applied in both experiments; dihydrocaffeic acid,

oleyl dihydrocaffeate, indicated better inhibition of volatile

development in Experiment 1 with acetone added for dihy-

drocaffeic acid emulsion, and in general no difference in the

inhibition percentages by oleyl dihydrocaffeate in the two

experiments. Hence, the addition of acetone seemed to have

no significant impact on the development of volatiles, which

was also concluded from the formation of peroxides. The

inhibition percentages indicate that three of the lipophilized

antioxidants were better than the phenolic they originated

from.

3.3.1 Tocopherol in the fish oil enriched emulsions

Three of the tocopherol homologues were detected (a-, g-

and d-tocopherol), but only the concentration of a-toco-

pherol changed significantly during the storage period

(Fig. 3). This might be due to the higher amount of this

homologue compared with the others. The concentration of

a-tocopherol was reduced most in the milk emulsion without

antioxidant added (control emulsion) and least in emulsion

with rutin laurate followed by oleyl dihydrocaffeate in

Experiment 1 and caffeic acid and oleyl dihydrocaffeate fol-

lowed by octyl dihydrocaffeate in Experiment 2. Hence,

tocopherol was better preserved in these emulsions maybe

due to less lipid oxidation.

3.3.2 Sensory evaluation

As volatiles cause off-flavour, the preliminary sensory evalu-

ation of emulsions was expected to reflect the result of volatile

development in the emulsions. Only milk emulsions in

Experiment 2 was evaluated by their odour by a sensory

expert panel at storage day 9 and 12 (data not shown).

Experiment 1 was not possible to evaluate due to addition

of the acetone, which resulted in a strong solvent odour.

Clearly, the most oxidized emulsion according to the sensory

evaluation was the emulsion with oleyl alcohol due to a clear

off-odour of oxidized fish oil at Day 9 which developed to

lacquer/painty at day 12.Moreover, the control emulsion had

a clear fishy off-odour as well as metallic odour at day 9. The

most oxidatively stable emulsions, as determined by odour

evaluation, were emulsions with oleyl and octyl dihydrocaf-

feate. These two emulsions were evaluated to have ocean and

chemical/flower odour, respectively. The expert panel could,

however, not discriminate between these emulsions with

respect to their level of fishy off-odour.

4 Discussion

4.1 Physical stability and lipid oxidation

All the milk emulsions were physically stable and no changes

in the droplet size during storage were observed. In

Experiment 1 there was a significant difference in size of

the lipid droplets in the control emulsion (0.45 mm) com-

pared to the other emulsions (0.59–0.74 mm). This could be

due to either less lipid incorporated in the emulsion or an

unintended slightly different homogenization than for the

other emulsions in this experiment. However, it is clear from

Table 2 that the emulsion had the same amount of fish oil as

the other emulsions. Thus, the smaller droplets may indicate

a slightly longer homogenization of this emulsion. Lipid

oxidation is initiated at the interface of the droplet, therefore

the size of total interfacial area is hypothesized to influence

the lipid oxidation. Thus, increased droplet size may result in

decreased lipid oxidation [9]. However, the literature in this

Figure 3. Concentration of a-tocopherol in the different fish oil

enriched emulsions at day 0 and 12 (A) Experiment 1 and (B)

Experiment 2. Error bars indicate SD of the measurements

(n ¼ 4). Different letterswithin same storage day indicate significant

differences in concentration betweenemulsions. Abbreviation: Rutin

C12, rutin laurate; Rutin C16, rutin palmitate; DHCA, dihydrocaffeic

acid; DHCA C18:1, oleyl dihydrocaffeate; DHCA C8, octyl

dihydrocaffeate.



area is conflicting, and some studies support the hypothesis

[4, 28], whereas in milk emulsions the opposite has been

observed [29, 30]. Therefore, the increased oxidation in the

control emulsion was most likely due to no protection of the

lipids by antioxidants and not due to the different droplet size.

Furthermore, the control emulsion in Experiment 2 had the

same droplet size as the other emulsions, but was still the

most oxidized emulsion together with emulsion with oleyl

alcohol. Hence, the results indicated limited effect of inter-

face area on lipid oxidation in complex emulsion systems.

4.2 Oxidative stability of the milk emulsions and theinfluence of alkyl chain length

The results indicated thatmilk emulsions without antioxidant

or with oleyl alcohol added generally oxidized faster than

emulsions with either phenolics or phenolipids added.

Interestingly, rutin laurate was a more efficient antioxidant

compared to rutin palmitate and rutin it-self, which indicated

a cut-off effect related to the alkyl chain length. However, for

lipophilized dihydrocaffeic acid it was less clear whether such

cut-off effect existed, as differences in oxidation parameters

not always was significantly different for octyl and oleyl

dihydrocaffeate emulsions.

In emulsion with rutin laurate, PV did not increase during

storage, which could indicate efficient inhibition by rutin

laurate or fast decomposition of peroxides to volatiles. The

findings that milk emulsion with rutin laurate added had low

concentrations of volatiles showed that, rutin laurate effec-

tively inhibited formation of both primary and secondary

oxidation products. Interestingly, the findings regarding

the efficacy of rutin esters as antioxidants were different in

milk emulsions compared with earlier findings in simple o/w

emulsion [13]. Thus, in this study on fish oil enriched milk,

both rutin esters had an antioxidative effect, but rutin laurate

was a more efficient antioxidant than rutin palmitate and

rutin it-self. In contrast, rutin laurate and rutin palmitate

were less effective antioxidants when compared with rutin

in simple o/w emulsion [13]. However, rutin laurate also

exerted stronger antioxidative activity than rutin and rutin

palmitate in a LDL assay, which is a more complex system

than an o/w emulsion [13]. Thus, these findings indicate that

the cut-off effect is influenced by the system, i.e. simple o/w

emulsion or more complex emulsion systems such as LDL

and milk. In addition, the fish oil enriched milk emulsion

contains proteins and otherminor components, which are not

present in a simple o/w emulsion. These components might

have interacted and influenced the efficacy or location of the

phenolipids. Shahidi and Zhong [15] suggested that not only

the partitioning of the antioxidant influence the efficacy of the

antioxidant, but also the emulsifier in emulsified medium.

This is due to saturation of the interfacial area by emulsifier,

which leaves less interfacial area available for antioxidant

location. Hence, emulsifiers may compete with antioxidants

for localization at the interface, where oxidation is initiated.

Moreover, recent experiments with phenolic compounds and

two different emulsifiers in simple o/w emulsion have shown

interaction of the antioxidants with emulsifier and iron [18].

The phenolic compounds investigated were caffeic acid,

coumaric acid, naringenin and rutin, which were evaluated

for interactions with Citrem and Tween with or without iron

present. Interactions in this study may have influenced the

efficacy and location of the antioxidant.

Both octyl dihydrocaffeate and oleyl dihydrocaffeate

exerted stronger antioxidative effects than dihydrocaffeic acid

in fish oil enriched milk. The efficacy of octyl dihydrocaffeate

was slightly better than oleyl dihydrocaffeate in inhibiting the

formation of some volatiles however for other volatiles it was

not significantly better. These findings are different from the

findings obtained with these phenolipids in a simple o/w

emulsion [17]. In the o/w emulsion system the difference

between octyl and oleyl dihydrocaffeate as antioxidants

was clearer than in the milk emulsions, where octyl dihydro-

caffeate was a significantly better antioxidant than oleyl

dihydrocaffeate. Importantly, in both studies oleyl dihydro-

caffeate contained impurities such as oleyl alcohol. When

added in the milk emulsion oleyl alcohol resulted in a proox-

idative effect in contrast to its addition in the simple o/w

emulsion. Since oleyl alcohol acted as a prooxidant in fish oil

enrichedmilk it might have reduced the antioxidative effect of

oleyl dihydrocaffeate as antioxidant. Thus, the antioxidative

effect may have been even better for oleyl dihydrocaffeate

than octyl dihydrocaffeate if a more purified compound was

used, but this needs to be further investigated. The slight

difference in results obtained from the o/w emulsion and milk

emulsion may, similar to effects of rutin esters, be explained

by the different systems tested. For example the effect of

emulsifier and its position on the interface may have led to

interactions with the antioxidant [15].

In vitro antioxidant assays preformed in our previous

studies have generally showed better antioxidant activity

for the original phenolic than for the lipophilized phenolic

compound [12, 17]. However, in emulsions the dihydracaf-

feates were better antioxidants than dihydrocaffeic acid in

both simple o/w emulsions and milk emulsions, and lipophi-

lized rutin was better than rutin in milk emulsions. Thus, the

results from in vitro antioxidant assays cannot solely predict

the antioxidant properties in emulsion systems, and other

factors such as interactions and partitioning may also influ-

ence antioxidant activity. The partitioning of lipophilized

dihydrocaffeic acid and rutin in the different phases has

previously been evaluated [13, 17]. Only rutin laurate could

be measured in the water phase of a simple o/w emulsion and

only in small amounts (3.8%). The finding that lipophilized

rutin did not have any antioxidative effect in o/w emulsions

compared to rutin, whereas lipophilization with C12

improved its efficacy in milk indicates that interactions with,

e.g. proteins may influence the location of the lipophilized

rutin in milk emulsions. Thus, rutin laurate may be located

more favourably in the milk emulsion to act as antioxidant,



i.e. close to the interface instead of as a micelle in the water

phase or in the core of the oil droplets. A similar explanation

could be given for the differences observed with oleyl dihy-

drocaffeate. However, further research is needed in order to

be able to conclude on possible interaction in the milk and

changed partitioning of the phenolipids as a consequence of

the interaction.

4.3 Fish oil enriched milk and human health

Overall it was possible to stabilize fish oil enriched milk by

adding phenolipids. The current amount of fish oil in this

milk will only be a supplement of n-3, since 500 mL milk

daily will result in an intake of 50 mg EPA and DHA. EU

recommendation is currently at an intake of 250 mg daily due

to proved health beneficial effects, thus 500 mL milk daily

will cover 20% of the recommendeted. Consumption of n-3

enriched milk solely may not increase the human health, but

together with other fish or fish oil enriched products.

5 Conclusions

In conclusion, both phenols and phenolipids acted as anti-

oxidants in milk emulsions enriched with fish oil. However,

the phenolipids were more efficient antioxidants especially

rutin laurate, octyl and oleyl dihydrocaffeate. Despite the fact

that only two chain lengths were evaluated, the results tend to

follow the cut-off effect in relation to alkyl chain length and

antioxidative effect for rutin esters. The optimal alkyl chain

length for a rutin ester in fish oil enrichedmilk is at least below

a chain length of 16 carbon atoms. For dihydrocaffeate esters

it was not possible to conclude on a specific cut-off effect in

relation to alkyl chain length and antioxidative effect. To be

able to conclude on the optimal lipid chain length attached to

the phenolic compounds in relation to their strongest anti-

oxidant protection, more studies on the effect of rutin and

dihydrocaffeic acid with different lipid chain lengths are

needed. The antioxidative effect of phenolipids seems quite

complex and it would be of great value to be able to under-

stand the effects of both the lipid chain length and the type of

emulsion system on the antioxidative effect of the lipophilized

compounds. Taken together, these results clearly show that the

polar paradox hypothesis is too simple and must be reconsid-

ered. Moreover, the almost untouched area of phenolipids as

antioxidants in real food systems deserves more attention as the

results have indicated very promising effects of these com-

pounds that could be utilized by the industry in the future.

We thank Maritex Norway (subsidiary of TINE BA, Norway)

for providing the fish oil to our research. The study was financed by

the Danish Council for Strategic Research (Programme committee

for food, nutrition and health) and the Directorate for food,

Fisheries and Agri Business.

The authors have declared no conflict of interest.

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